This highly innovative project consists of four aims and investigates the dynamics of eukaryotic nucleotide excision repair proteins at the single molecule level. Specifically, this study analyzes DNA damage through five discrete steps involving: i) initial non-target DNA binding by a NER recognition complex; ii) diffusion of this repair complex to a lesion site; iii) lesion processing, conformational proof reading by this complex (3) (such as insertion of a beta-hairpin into the DNA (Rad4/XPC); iv) arrival of a DNA repair recognition complex ; and v) lesion processing by the second repair complex causing the first repair complex to diffuse away from the damaged site. We hypothesize that the efficiency of the hand-off from one protein complex to the next is the rate-limiting step(s) in NER. Aim 1 investigates the interactions of purified Rad4-Rad23-Rad33 or XPC- RAD23B-CETN2 complexes with damaged DNA. The second aim measures the kinetic interactions of Rad14 or XPA with damaged DNA. The particularly original third aim studies the interaction of specific NER damage recognition components in whole-cell extracts of Saccharomyces cerevisiae. Genetic ablation or site-directed mutation into specific genetic loci will allow analysis of the important domains that are essential for damage recognition and lesion hand-off. The fourth aim measures the damage handoff from UV-DDB to XPC-RAD23B and XPA and how ubiquitylation or PARylation stimulates this process. This project will give an unprecedented view of the complex process of damage recognition steps of eukaryotic nucleotide excision repair and answer several key questions regarding damage recognition that have been intractable in the absence of single molecule approaches. Completion of this project will have a long and lasting impact on the field.